Nephrotoxins remain a major cause of acute kidney injury (AKI). RNA interference holds great promise as a novel and specific therapeutic gene-silencing technology for a wide range of diseases. Oligonucleotides, like many other small macromolecules, are present in the ultrafiltrate formed at the renal corpuscle, and therefore are presented to the tubular epithelial cells lining the nephron at relatively high concentrations. These cells, particularly those in the proximal tubule segments, avidly take up oligonucleotides, so that the kidney has by far the greatest accumulation of systemically delivered oligonucleotides. This makes siRNA a promising technology for treatment of kidney disease, especially given the particular sensitivity of proximal tubule cells to toxic or ischemic injury. Given the clinical and biological importance of these observations, the handling of siRNA by renal cell types needs to be better characterized. We propose that siRNA is preferentially accumulated in proximal tubule epithelial cells because of the specialization of these cells for rapid endocytosis of macromolecules delivered to the filtrate. New technologies in fluorescence microscopy, particularly the application of multi-photon imaging to intravital microscopy, allow the cellular and subcellular distribution of labeled oligonucleotides to be analyzed in real time in live animals. We propose to use this methodology in four specific aims to better understand the mechanism of uptake of siRNA by proximal tubule cells and to establish the feasibility of using siRNA to prevent toxic acute kidney injury (AKI). We propose: 1) to determine the kinetics of cellular and intracellular accumulation, catabolism and biological effects of siRNA in the normal kidney;2) to determine the effect of chronic kidney disease on siRNA handling by the kidney;3) to determine the effect of endocytic blockade on siRNA uptake;and 4) to test the ability of siRNA to prevent aminoglycoside uptake by proximal tubule cells and hence limit renal toxicity. PUBLIC HEALTH RELEVANCE: RNA interference is a method that can be used to stop the harmful effects of normal or mutant proteins, and may be an important and specific therapy for diseases in the future. The RNA molecules used in this technology are concentrated in the kidney, and the studies in this proposal will examine the mechanisms and consequences of this accumulation, the way that kidney disease can affect this process, and the possibility of using RNA to protect the kidney against toxins.